Background
Dengue fever (DF), an ancient disease with a history of approximately 2000 years, is caused by four different but closely related dengue viruses (DENV-1, DENV-2, DENV-3, and DENV-4) and is mainly transmitted by female
Aedes aegypti or female
Aedes albopictus [
1,
2]. DF occurs in tropical and subtropical urban and semi-urban areas around the world. The global dengue epidemic has spread quickly in recent decades from 9 endemic countries before 1970 to 128 in 2012 [
3]. The incidence of dengue has also increased dramatically from 1.2 million in 2008 to 3.9 million in 2015 [
4,
5]. In 2016, there were more than 2.38 million cases in the Americas alone, of which Brazil accounted for approximately 1.5 million cases. Meanwhile, more than 375,000 cases were reported in the Western Pacific region, including 176,000 cases in the Philippines and 100,000 cases in Malaysia [
6]. However, the number of dengue cases reported in the Americas in 2017 was 580,000, which was approximately 78.9% fewer than the previous year. According to data provided by the World Health Organization (WHO), the number of cases in the first quarter of 2018 decreased by 27% compared with that in the same period of 2017; during this period, DF cases were mainly reported in countries such as Paraguay, Argentina, Bangladesh, Cambodia, India, Myanmar, Malaysia, Pakistan, Thailand, Yemen, and China and were mainly caused by the DENV-1 and DENV-2 serotypes [
7].
DF has become a serious public health problem in China. According to data provided by the Chinese Center for Disease Control (CCDC), 757,243 people have been infected in the past 42 years [
8,
9], and these infections largely occurred in Hainan [
10], Guangdong [
11,
12], Zhejiang [
13,
14], Fujian [
15], Taiwan, and Yunnan [
16‐
19]. In 2018, an unexpected dengue outbreak occurred for the first time in Hunan Province, an inland province of China. According to data provided by the Centers for Disease Control and Prevention (CDC), the earliest DF case was reported on September 2. On October 6, 172 infected individuals were confirmed as NS1-positive, with one death in Hunan Province. Qiyang County was the location most seriously affected by the epidemic; 73 cases were confirmed in Qiyang County from September 8 to September 14, accounting for 76.04% of the total confirmed cases in this area. The ratio of female to male infected patients was 1.04 to 1 (49:47), with an average age of 49.5 years (ranging from 11 to 84 years old). It should be noted that no dengue cases were found in this area from 2000 to 2013; only five imported cases were reported between 2014 and 2017, and no local cases were reported in Qiyang County.
This was the first dengue outbreak in Hunan, an interior province of China. This outbreak provides us with an early warning that dengue fever has gradually spread inland from China’s coastal and border regions and highlights the urgent need to monitor the cross-border and cross-regional spread of dengue virus. The purpose of this study was to verify the causative agent and analyse the molecular characteristics of the epidemic strain in this outbreak.
Discussion
In mainland China, dengue fever mainly occurs in Guangdong, Hainan, Zhejiang, Fujian, Guangxi, and other coastal regions or in Yunnan Province and Southeast Asian countries adjacent to Yunnan Province. Only scattered cases have been reported in inland China, but no large-scale dengue epidemic has been reported in the inland area to date. Hunan is an inland province of China located near 30 degrees north latitude. The climate is warm and humid from June to November, which provides a natural environment for the breeding of Aedes albopictus. Hunan Province is located near Guangdong, Guangxi, Zhejiang, and other areas with a high incidence of dengue fever. The total number of dengue infections in China was 5106 in 2018, which included 3250 cases in Guangdong Province, 217 cases in Zhejiang, and 172 cases in Hunan. This was the first dengue outbreak in Hunan, an inland province of China, which provides us with an early warning that dengue fever has gradually spread from coastal and border areas to inland areas of China and highlights the urgent need to monitor the cross-border and cross-regional transmission of dengue viruses.
In this study, we collected serum from 260 patients with dengue fever in Qiyang County, Hunan Province, and 96 of the cases were confirmed to be NS1-positive. Of them, 89 viral RNAs were extracted, and structural protein gene fragments (HNQY2018001–089) were obtained by amplification of overlapping fragments with a length of 2325 nucleotides. Phylogenetic tree analysis showed that all isolated strains were of the cosmopolitan DENV-2 genotype, belonged to one cluster of the ML tree and were closely related to the Zhejiang strain (2017, MH110588). Additionally, the isolated strains were closely related to strains from Malaysia (KJ806803, 2013), Bali (KT806318, 2014), Indonesia (KT781561, 2014) and the Philippines (KU517847, 2015). Although all four serotypes of dengue virus were prevalent in Zhejiang Province in 2017, the vast majority of the epidemic strains were still of the cosmopolitan DENV-2 genotype, and the encoded protein of this epidemic strain has only one amino acid that differs from that of the epidemic strain in Hunan Province in 2018. This result suggested that the DENV-2 epidemic in Hunan was possibly imported from Southeast Asian countries, such as Malaysia, Indonesia or the Philippines, passed through Zhejiang Province, and then spread to Hunan Province.
Compared with that of the standard strains, 17 amino acid substitutions were observed in the structural protein C/prM/E of all 89 epidemic strains. The prM-E protein is the main structural protein of flaviviruses and is related to virulence, host affinity, virus adsorption, penetration, and cell fusion [
27]. Hydrophobic amino acids play an important role in maintaining the tertiary structure of proteins due to their hydrophobic interactions and may impact the virulence of the virus. Tamm et al. found that hydrophobic domains affect the virulence potential of Yersinia enterocolitica [
28]. Sainz et al. determined that single hydrophobic amino acids play an important role in transcriptional activation in vivo [
29]. In our study, three hydrophobic amino acids in the CDS region were mutated into hydrophilic amino acids, namely, the 196th (M82: T → A), 262nd (M148: H → Y), and 351st (E71: D → A) amino acids. In addition, a neutral amino acid became a basic amino acid at the 332nd (E52: Q → H) position, and two positively charged amino acids were converted into negatively charged amino acids at the 406th (E126: K → E) and 429th (E139: H → N) positions. Mutations in these amino acids have not been reported, and changes in polarity or charge of amino acids may affect the functions of the prM and E proteins; however, further studies are needed to confirm these hypotheses. DENV E protein domain III (E295 ~ E395) undergoes immunoglobulin G (IgG)-like folding and plays an important role in mediating the fusion of viruses and host receptors [
30]. In this study, there were two amino acid changes in the EDIII domain at the 602nd (E322: I → V) and 670th (E390: N → S) positions. It has been reported that the mutation of E390 from asparagine to serine can enhance the replication ability of viruses [
31], but the influence of the E322 amino acid mutation remains to be determined.
Changes in protein secondary structure will affect enzyme activity. Compared with the DENV-2 standard strain (DENV2-SS), the isolated strains lacked eight protein binding sites (4, 5, 122, 120, 133, 584, 596, and 642) and two polynucleotide binding sites (6 and 18). Moreover, four new protein binding sites (19, 29, 144, and 377) and one polynucleotide binding site (74) emerged in the isolated strains. Furthermore, approximately eight obvious changes were observed in the buried and exposed regions. All of the above changes may lead to the diversification of protein structural domains and further influence protein function. Homologous modelling and prediction of the possible 3D structures of structural proteins showed that the structural proteins from the epidemic strains and DENV2-SS had similar 3D structures and shared 4 predicted protein binding sites, and only one protein binding site (429th) differed between them (DENV2-SS: HIS429; Zhejiang/2017 and Hunan epidemic strain: ASN429).
The analysis showed that there were no recombination events between the Hunan epidemic strains and the 111 DENV-2 reference strains, and no distinct positive selection sites were detected in the structural proteins, which contained 775 amino acids, which suggests that these structural protein-coding genes were conserved.
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